Fabrication of

Polyhydroxyalkanoates
(PHA) Bioplastics from
Sugarcane Bagasse and
butylated hydroxyanisole for
Color Changing Food
Packaging Production
 ABSTRACT

Food packaging is a critical step in the food manufacturing process because it

protects the food from infection, maintains its quality, and extends its shelf life.
However, poor food packaging may result in reducing its freshness and spoilage that
leads to food waste. The purpose of this study is to analyze how the incorporation of
sugarcane bagasse and butylated hydroxyanisole affects the physical and chemical,
mechanical, thermal, biodegradability, and surface of producing the
Polyhydroxyalkanoates bioplastic. All of the ingredients consisting of 1.5 g of bagasse
fibres, butylated hydroxyanisole at concentrations of 3 wt. %, dissolved 50 mg PHA
extract in 10 ml chloroform 1 mL of glycerin, 40 mL of distilled water, and 0.5 g sorbitol
will be prepared and will be blended in a 250 mL beaker. Significant color changes in
dry films as a function of pH will be detected, indicating a promising approach for their
usage as intelligent sensors in food packaging. The researcher will produce an efficient
Polyhydroxyalkanoates bioplastic from sugarcane bagasse and butylated
hydroxyanisole for color-changing food packaging. This study aims to benefit the
society, and it will reduce the food spoilages by giving a warning to consumers about
the conditions inside the food package through a color changing food packaging.

Keywords: Polyhydroxyalkanoates, sugarcane bagasse, butylated hydroxyanisole,

color changing, bioplastic
 TABLE OF CONTENTS

Chapter 1: Introduction Page #

Background of the Study 1
Statement of the Problem 3
Statement of the Objectives 3
Statement of the Hypotheses 4
Significance of the Study 5
Scope and Limitations 6
Chapter 2: Review of Related Literature and Studies Page #
Related Literature 7
Related Studies 9
Chapter 3: Methodology Page #
Collection of Materials 14
Isolation, selection and Optimization of bacterial growth of PHA- 14
producing bacteria from Sugarcane Bagasse
Production of PHA by selected isolates 14
Measurement of Dry Biomass 15
Extraction Process of PHA 15
Purification Process of PHA 15
Fibre extraction from sugarcane bagasse 16
Production of Bioplastic Film 16
Characterization of Film: Film Thickness 17
Characterization of Film: WVP Measurements 17
Characterization of Film: Mechanical Properties 17
Characterization of Film: Biodegradability of bioplastic 18
Characterization of Film: Film Color 18
Characterization of Film: FTIR Analysis 18
Characterization of Film: Total Phenolic Content 19
Characterization of Film: Antioxidant Activity of the Films 19
Statistical Analysis 20
Bibliography 21
Research Plan 23
Appendices 32
 CHAPTER 1

INTRODUCTION

Background of the Study

Food packaging is a critical step in the food manufacturing process because it

protects the food from infection, maintains its quality, and extends its shelf life.

However, it is responsible for polluting our climate, blocking our drains and potentially

harming wildlife when it ends up in our oceans and rivers. The Philippines has the

highest volume of uncontrolled plastic waste among Southeast Asian countries in a

2015 research by Ocean Conservancy and McKinsey Center for Business and

Environment.

Food packaging accounted for around 79% of branded plastic residual trash,

with home and personal care goods accounting for 12 and 8%, respectively.1 Poor

food packaging may result in reducing its freshness and spoilage that leads to food

waste. 280,000 tons of food waste with an economic value of $882 million was

recorded due to poor food packaging.2

When compared to petroleum-based alternatives, PHAs are more costly. Thus,

previous studies suggested that PHAs can be employed in biocomposite materials

including bio-based agro-residues to reduce costs while preserving performance in

some applications. Organic additives and fibers made of cellulosic material can help

polymers perform better. They boost biodegradation rates in all settings when

employed in biocomposites with PHAs.

1
David Bodamer. “EPA’s Latest MSW Estimates.” Waste460, November 16, 2016.
https://www.waste360.com
2
Ramos, Marjaleen. “17.5 billion of plastics every year.” Manila Bulletin. January 2020.https:mb.co

1
 Bagasse is the waste from sugar cane plants left over after the sugar has been

extracted; sugarcane bagasse will be utilized as a fiber source in the bioplastic

production.4 Sugarcane bagasse fiber reinforced polymer composites help to improving

advantages such as being ecologically beneficial materials at the production,

processing, and disposal stages. 5

Butylated hydroxyanisole (BHA) is white waxy solid that is soluble in fat and

become transparent. It is inexpensive, efficient, and highly stable under normal

processing conditions. BHA is particularly effective as an antioxidant in animal fats, but

less so in vegetable oils and may be the best plasticizer and antioxidant for polymers. 6

Therefore, this study aims to determine the performance of the color-changing

activity of Polyhydroxyalkanoates bioplastic from Sugarcane bagasse and Butylated

Hydroxyanisole. This study’s purpose is to reduce the food spoilages by giving a

warning to consumers about the conditions inside the food package through a color

changing food packaging.

___________________________________________________
4
Azmin Siti, et al. ‘Development and characterization of food packaging bioplastic film from
cocoa podhusk cellulose incorporated with sugarcane bagasse fiber.” Journal of Bioresources
and Bioproducts. November 2020.
https://www.sciencedirect.com/science/article/pii/S2369969820301146?via%3Dihub
5
Sundarraj Chockalingam, et al. “Bagasse Fiber – The Future Biocomposite Material: A
Review,” International Journal of ChemTech Research. January 20, 2014.
https://www.researchgate.net/publication/270453697_Bagasse_Fiber_-
_The_Future_Biocomposite_Material_A_Review
6
Fujita Shuzo, et al. “Function of Some Antioxidants as a Plasticizer for the Biodegradable
Plastic, Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P-(HB-HV)].” Journal of Home
Economics of Japan. January 21, 2021.
https://www.jstage.jst.go.jp/article/jhej1987/54/3/54_3_193/_pdf/-char/ja
Statement of the Problem

This study aims to analyze how the incorporation of sugarcane bagasse and

2 and chemical, mechanical, thermal,

butylated hydroxyanisole affects the physical
biodegradability, and surface of producing the Polyhydroxyalkanoates bioplastic. Thus,

the researcher wants to ask the following questions:

 Does increasing the amount of Sugarcane Bagasse affect the strength of

the color-changing food packaging?

 Does increasing the amount of butylated hydroxyanisole affect the intensity

of color changing of the food packaging?

 Does the cellulose in Sugarcane Bagasse affect the color-changing activity of

Polyhydroxyalkanoates bioplastic?

Statement of the Objectives

This study aims to fabricate Polyhydroxyalkanoates bioplastic from sugarcane

bagasse and butylated hydroxyanisole. Thus, these are the specific objectives of the

researcher:

 To isolate, collect, and purify bacteria from sugarcane trash.

 To produce PHA from the selected isolates.

 To measure the dry biomass.

 To extract PHA from the biomass via reflux.

 To purify the extracted PHA with 1-butanol via reflux.

 To extract fibres from the Sugarcane bagasse.

 To produce a Polyhydroxyalkanoates bioplastic film from sugarcane bagasse

and butylated hydroxyanisole.

 To determine the physical properties of Polyhydroxyalkanoates bioplastic from

sugarcane bagasse and butylated hydroxyanisole by its film thickness.

 To measure the WVP measurement of the PHA bioplastic by Differential relative

3
 humidity (RH).

 To investigate the mechanical properties of sugarcane bagasse and butylated

hydroxyanisole using tensile testing machine.

 To determine the degradation of Polyhydroxyalkanoates bioplastic by placing

Aspergillus niger.

 To measure the film color of the PHA bioplastic with a colorimeter using the CIE-

Lab color scale.

 To determine the molecular level modifications induced by the incorporation of

BHA into the polymer chains using the FTIR spectrometer.

 To evaluate the color response of the Polyhydroxyalkanoates bioplastic that

isexposed to different pH.

 To investigate the Antioxidant activity and total phenolic content using a

modified ferric reducing antioxidant power (FRAP) method.

Statement of the Hypothesis

This study hypothesizes the following:

 If the Sugarcane bagasse is related to tensile strength, then the higher amount

of it will make the packaging more durable.

 If the butylated hydroxyanisole is related to anthocyanin, then the intensity of

color-changing will increase and ensure the shelf-life of foods inside the

packaging.

 If the cellulose of Sugarcane Bagasse has high antioxidant, then that affects the

color-changing activity of Polyhydroxyalkanoates

4 bioplastic.
Significance of the Study

This study makes a significant contribution to the immediate need to reduce

food waste by limiting the use of non-biodegradable plastic in food packaging and

replacing it with more environmentally friendly materials that increase the shelf life of

food. It aims to benefit the society, and it will reduce the food spoilages by giving a

warning to consumers about the conditions inside the food package through a color

changing food packaging.

Bioplastics such as polyhydroxyalkanoates (PHAs) are considered an ideal

candidate for food packaging applications. PHAs are microbiologically produced

polyesters with tunable mechanical and physical properties, have high biodegradability

and biocompatibility; therefore they are an environmentally friendly material to use in

home and workplace. Its promising features include biodegradability, hydrophobicity,

nontoxicity, thermoplasticity, and impermeability to gases and/or water.7 Sugarcane

bagasse wastes are chosen as an ideal raw material in manufacturing new products

because of its low fabricating costs and high quality green end material.8 Bagasse fiber

is obtained from a source which is known for its renewability in terms of fast growth

and better mechanical properties.9 Butylated Hydroxyanisole is a phenolic compound

that is added to foods to preserve fats.

7
F. Masood. “Polyhydroxyalkanoates in the Food Packaging Industry.” Nanotechnology
Applications in Food. 2017.
https://www.sciencedirect.com/science/article/pii/B978012811942600008X?via%3Dihub
8
Sundarraj Chockalingam, et al. “Bagasse Fiber – The Future Biocomposite Material: A
5
Review,” International Journal of ChemTech Research.
9
Ibid.
Scope and Limitations of the Study

This study is to produce polyhydroxyalkanoates using sugarcane bagasse and

butylated hydroxyanisole as an active component in food packaging. This study did not

involve testing its effectiveness on food storage and preservation, and although related

studies have confirmed its success in prolonging shelf life.

Sugarcane bagasse is the fibrous remains of the sugarcane stalk once it has been

crushed. Production of sugarcane bagasse was not a part of this study, and this was readily

available in fiber form. The researcher obtained the sugarcane bagasse from the Suki Market

in Quezon City. The butylated hydroxyanisole that is often added to foods to preserve fats and

oils will be purchased from UNAHCO, Inc. in Mandaluyong.

The experimental groups in this study are: sample PHA bioplastic film A has both equal

amount of sugarcane bagasse fiber and butylated hydroxyanisole. Sample PHA bioplastic film

B has greater amount of sugarcane bagasse fiber than butylated hydroxyanisole. Sample

PHA bioplastic film C has greater amount of butylated hydroxyanisole than sugarcane

bagasse fiber. The concentrations used were 75꞉25, 50꞉50, and 25꞉75 of sugarcane bagasse

fiber to butylated hydroxyanisole ratio and vice versa. The control group is the PHA bioplastic

film without the BHA and fibre.

The other materials or chemicals will be obtained from different laboratories in Metro

Manila. The methods and procedures will be conducted in different laboratories that offer

WVP measurements, pH testing, tensile testing machine, colorimeter, FTIR Analysis, Folin-

Ciocalteu Method, FRAP method, and DSC Analysis. This study will take approximately two

months to complete, including the preparation of materials.

6
 CHAPTER 2

REVIEW OF RELATED LITERATURE AND STUDIES

RELATED LITERATURE

Polyhydroxyalkanoates

PHAs can be used to make completely compostable, soil, and marine

biodegradable products – a significant advantage when compared to the negative

consequences of plastic landfilling. PHAs can be employed in biocomposite materials

including bio-based agro-residues to lower costs while preserving performance in

specific applications. Organic fillers and fibers made of cellulosic material can increase

the characteristics of polymers; nevertheless, their influence on the marine

biodegradability of the composite matrix is still unknown. They boost biodegradation

rates in all settings when employed in biocomposites with PHAs.1 PHAs are the most

versatile fully biodegradable polymers with properties similar to conventional plastics.2

Sugarcane Bagasse

Sugarcane bagasse is the crushed cane after the juice from sugarcane is being

extracted. Both wastes were commonly thrown away without knowing the benefits of

transforming it into bioplastic or anything else.3 SCB wastes are chosen as an ideal raw

1
Amar Mohanty, et al. “Review of recent advances in the biodegradability of polyhydroxyalkanoate
(PHA)
bioplastics and their composites.” Green Chemistry. August 17, 2020.
https://pubs.rsc.org/en/content/articlelanding/2020/gc/d0gc01647k
2
Ipsita Roy, et al. “Polyhydroxyalkanoates: bioplastics with a green agenda.” Current Opinion in
Microbiology. June 2010. https://doi.org/10.1016/j.mib.2010.02.006
3
Azmin Siti, et al. ‘Development and characterization of food packaging bioplastic film from cocoa pod
husk cellulose incorporated with sugarcane bagasse fiber.” Journal of Bioresources and Bioproducts.
November 2020. https://www.sciencedirect.com/science/article/pii/S2369969820301146?via%3Dihub

7
material in manufacturing new products because of its low fabricating costs and high

quality green end material. It is ideal due to the fact that it is easily obtainable given the

extensive sugar cane cultivation making its supply constant and stable.4

Butylated Hydroxyanisole

Butylated hydroxyanisole (BHA) is a phenolic molecule commonly used in foods

to preserve fats and oils and protect them from going rancid. It is used in food,

cosmetics, and the packaging of fat-containing items to preserve nutritional levels, color,
5
flavor, and odor. BHA is a mixture of the isomers 3-tert-butyl-4-hydroxyanisole and 2-

tert-butyl-4-hydroxyanisole. Also known as BOA, tert-butyl-4-hydroxyanisole, (1,1-

dimethylethyl)-4-methoxyphenol, and tert-butyl-4-methoxyphenol. Its molecular formula

is C11H16O2 and a white or yellowish waxy solid. It also has faint characteristic aromatic

odor.6

Anthocyanin

Anthocyanins are colored water-soluble pigments belonging to the phenolic

group. Anthocyanins responsible for the colors, red, purple, and blue, are in fruits and

vegetables, depending on their pH. 7 Anthocyanins are well-known alternatives to

4
Sundarraj Chockalingam, et al. “Bagasse Fiber – The Future Biocomposite Material: A Review,”
International Journal of ChemTech Research. January 20, 2014.
https://www.researchgate.net/publication/270453697_
5
Anne Helmenstine. “Chemistry of BHA and BHT Food Preservatives.” ThoughtCo. July 30, 2019.
https://www.thoughtco.com/bha-and-bht-food-preservatives-607393
6
Adanze Nwakaudu, et al. “The Use of Natural Antioxidant Active Polymer Packaging Films for Food
Preservation.” AppliedSignals Research & Publications. November 2, 2015.
https://www.researchgate.net/publication/290821241/
7
Azrina Azlan, et al. “Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical
ingredients, and the potential health benefits.” Food and Nutrition Research. August 13, 2017.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613902/

8
synthetic dyes.8 Incorporating anthocyanin into food package matrices applies the bio-

switch concept, which aids automatic response to changes (external stimuli) in the

environment. 9

Antioxidant Activity

Antioxidants are additives commonly used in the polymer industry to prevent

thermal degradation of polymers during processing.10 Antioxidant packaging materials

are being developed by incorporating the active compounds in the polymer matrix or on

the polymeric film surface. The antioxidant activity of the material is based on a

migration process into the food; the substances released should be food additives and

comply with present regulations in terms of maximum concentration. 11

RELATED STUDIES

Development and Characterization of Food Packaging Bioplastic Film from Cocoa

Pod Husk Cellulose Incorporated With Sugarcane Bagasse Fibre

(Azmin Siti, et al. 2020)

Agricultural wastes like cocoa pod husk and sugarcane bagasse are becoming

more common. The development of food packaging biofilms from these two wastes

might benefit both the environment and humans. As a result, this study was conducted

in order to develop biodegradable plastic films using cocoa pod husk and sugarcane

8
Muhammad Oka Ramadhan, et al. “Anthocyanins from Agro-waste as Time-Temperature Indicator to
Monitor Freshness of Fish Products.” Indonesian Journal of Educational Research and Technology.
May17, 2021. https://ejournal.upi.edu/index.php/AJSE/article/view/34228/14758
9
N H C Hamzah,et al. “Smart food packaging from sago starch incorporated with anthocyanin from
Brassica oleracea.” IOP Conf. Ser.: Earth Environ. 2021. https://iopscience.iop.org/article/10.1088/1755-
1315/733/1/012030/pdf
10
Ramon Catala, et al. “Advances in antioxidant active food packaging.” Trends in Food Science &
Technology. January 2014. https://www.sciencedirect.com/science/article/abs/pii/S0924224413002288
11
Jonas Contiero, et al. “Polyhydroxyalkanoate Synthesis by Burkholderia glumae into a Sustainable
Sugarcane Biorefinery Concept.” Bioprocess Engineering. January 13, 2021.
https://www.frontiersin.org/articles/10.3389/fbioe.2020.631284/full

9
bagasse. To extract cellulose and fiber, cocoa pod husk and sugar cane bagasse were

utilized, respectively. The bioplastic sheets generated were split into several cellulose

and fiber concentration ratios, including 1000 (100 percent cellulose), 7525 (cellulose

fibre), and 0100 (cellulosefibre) (100 percent fibre). Sensory evaluation, drying time,

moisture content, water absorption, and water vapor permeability were all evaluated for

each bioplastic concentration ratio.

Characterization of Food Packaging Films with Blackcurrant Fruit Waste as a

Source of Antioxidant and Color Sensing Intelligent Material

(Mia Kurek, et al. 2021)

Reduce food production losses while improving the functional features of

produced films aimed towards food coatings and wrappers. As a bio-based product,

chitosan and pectin films were augmented with blackcurrant powder. WVP of active

films rose by up to 25%, whereas moisture content increased by 27%. The presence of

insoluble particles largely lowered mechanical properties. DSC tests indicated that the

degradation temperatures for chitosan-based samples were reduced by 18 °C and those

for pectin-based samples containing blackcurrant powder were reduced by 32 °C,

indicating a disruption in polymer stability. The dried films' brightness and redness

varied substantially depending on the polymer type. Significant color changes were

seen after being exposed to different pH buffers, notably in chitosan film formulations.

10
This effect is being studied further in formulations that will be employed as color change

indicators in intelligent bio packaging.

Utilization of Cellulosic Waste from Tequila Bagasse and Production of

Polyhydroxyalkanoate (PHA) Bioplastics by Saccharophagus degradans

(Mark Riley, et al. 2008)

Because of environmental concerns, the use of agricultural waste is becoming

increasingly significant. The objectives of this study were to assess the capabilities of a

rare organism, S. degradans, to degrade the primary components of plant cell walls and

to assess S. polyhydroxyalkanoates are produced by degradans. The expansion of S.

degradans was tested in MM containing glucose, cellobiose, avicel, and bagasse, and

all were shown to be capable of supporting growth. Cells were able to connect to

bagasse fibers; however, growth on these insoluble fibers was substantially slower,

resulting in lower maximum biomass output than with simple sugars. This study shows,

for what we believe for the first time, that a single organism can breakdown insoluble

cellulose while both producing and accumulating PHA under identical circumstances.

More research is needed to completely describe these capabilities and enhance PHA

synthesis and purification.

11
 Smart food packaging from sago starch incorporated with anthocyanin from

Brassica oleracea

(N H C Hamzah, et al. 2021)

Any material or method used to detect, perceive, and record any changes during

the life cycle of packaged goods or their surroundings is referred to as smart packaging.

The pH indicator film produced from sago starch and anthocyanin derived from red

cabbage was used in this work. Incorporating anthocyanin into food packaging matrices

employs the bio-switch principle, which assists in automatic reaction to environmental

changes. The color variations of the bio-switch system inform consumers to the

freshness of the food. The color of the sago starch-based film with anthocyanin varied in

response to pH, and SEM revealed the compatibility of the starch with the anthocyanin

extract as well as the film shape. When compared to the control film, FTIR chemical

analysis of the produced films revealed no chemical alterations. Smart food packaging

is a promising solution to customers' need for packaging that supports a hectic lifestyle

while also providing more economic possibilities.

Anthocyanins from Agro-waste as Time-Temperature Indicator to Monitor

Freshness of Fish Products

(Muhammad Oka Ramadhan, et al. 2021)

Microbial contamination causes fish items to spoil quickly. Every 10°C rise in

temperature causes an increase in the rate of microbial activity. The time-temperature

indicator (TTI) with variations in pH is one indication that may identify food quality;

however, these indicators are typically constructed of synthetic chemicals that

contaminate the environment. The purpose of this study was to review previous

12
research on anthocyanins from agro-waste as a time-temperature signal for monitoring

the freshness of fish products. This study employed a systematic literature review (SLR)

from a variety of relevant studies and resources. The SLR process involves practical

screening, quality assessment, synthesis, and review. Purple yam peel, black plum

peel, blueberries peel, rambutan peel, sweet potato peel, and dragon fruit peel are

anthocyanins obtained from agro-waste that may be turned into TTI. The TTI derived

from agro-waste anthocyanins has the potential to be used to check the freshness of

seafood items.

13
 CHAPTER 3

METHODOLOGY

1. Collection of Materials

1 kilogram of Sugarcane bagasse will be collected from a sugarcane juice that

was readily available from the Suki Market in Quezon City for consumption while 100

ml of butylated hydroxyanisole will be purchased from UNAHCO, Inc. in Mandaluyong.

2. Isolation, selection and Optimization of bacterial growth of PHA-

producing bacteria from Sugarcane Bagasse

Bacteria will be isolated, will be collected, and will be purified from sugarcane trash

as well as reference isolates known to produce bioplastics (e.g. Ralstoniaeutropha,

Azospirillumbrasilense, Rhizobiumsp, Bacillus sp) will be evaluated for their ability to

produce bioplastics. Different nutrient media are being evaluated in shake-flask culture and

under different conditions of shaking and incubation temperature so as to optimize the bulking

of bacterial cells.

3. Production of PHA by selected isolates

Mineral salts medium (MSM): Urea, Yeast extract, KH2PO4, Na2HPO4,

MgSO4∙7H2O, CaCl2, Glucose, and trace element solution 0.1 ml will be used for the

production of PHA by the selected isolates. Both glucose and trace element solution

will be autoclaved separately, and reconstituted prior to inoculation. The culture will

be prepared by sub culturing the isolates twice in nutrient broth. Then one ml of a 24 h

old culture will be inoculated into 100 mL production medium and will be incubated at

37 °C and 150 rpm for 48 h.

14
4. Measurement of Dry Biomass

For dry biomass measurement the culture will be centrifuged at 10,000 rpm for 15

min, and the pellet will be dried in an oven at 55 °C to constant weight.

5. Extraction Process of PHA

The PHA will be extracted from the biomass via reflux by adding 25 mL of dimethyl

carbonate (DMC) as a solvent to different amounts of biomass in a flask connected to a

cooling column. Biomass to solvent ratios, 0.01 g mL−1, 0.05 g mL−1, and 0.1 g mL−1 will

be tested in duplicate by adjusting the amount of biomass. This biomass to solvent

ratios will be referred as 1%, 5%, and 10%, respectively. The flask is immersed in a pan

filled with glycerin, previously heated up to the boiling point. Different times of extraction

will be tested (1 h, 1.5 h, and 2 h). After the extraction, the pan will be removed to cool

down to room temperature. A vacuum filtration with filter paper will be used to separate

the biomass from the solution. A rotary evaporator will be used to recover the solvent

and to separate the PHA in the form of a film attached to the wall of the flask. The

obtained PHA will be left to dry overnight and then will be weighed.

6. Purification Process of PHA

The extracted PHA will be purified with 1-butanol via reflux. 20 mL of the solvent will

be added to different amounts of the polymer in a flask which will be connected to a

cooling column. The different PHA to solvent ratios tested in duplicate will be 0.02 g

mL−1 and 0.04 g mL−1. The flask was immersed in a pan with glycerin and heated up to

the boiling point of 1-butanol. The purification times will be tested are 1 h and 2 h. The

flask will be taken from the pan, closed with a cap and will be allowed to cool down at

room temperature overnight. The solvent will be separated from the jelly-like

polymer by physically pressing it out with a cloth. The PHA will be allowed to dry at

15
room temperature overnight. The 1-butanol will be separated from the solubilized

contaminants and will be recycled with a rotary evaporator. The experimental data will

be obtained under the different purification conditions were compared using first an F-

test for variances and then the adequate t-test for the p values.

7. Fibre extraction from sugarcane bagasse

Firstly, 100 g of fine bagasse powder will be treated with 1000 mL of NaOH solution

for an hour with constant stirring at room temperature. After that, the treated bagasse

solution will be washed and filtered using distilled water until a neutral pH will be

obtained. The extracted fibre will be dried in an oven at 50 °C for 24 h. Then, 25 g of the

dried fibre will be bleached by stirring it with a 200 mL solution containing 1 mL of

vinegar and 3 g of NaCl on the hot plate for two hours at 70 °C. The bleached fibre will

be oven-dried at 50 °C for 24 h. This bleaching process will allow the fibres to be less

susceptible to UV and moisture, which will be considered as a factor for the degradation

process. The dried and bleached fibres will be kept for further use after grinding and

sieving procedures.

8. Production of Bioplastic Film

All of the ingredients consisting of 1.5 g of bagasse fibres, butylated hydroxyanisole

at concentrations of 3 wt. %, dissolved 50 mg PHA extract in 10 ml chloroform 1 mL of

glycerin, 40 mL of distilled water, and 0.5 g sorbitol will be prepared and will be blended

in a 250 mL beaker. After that, the solution will be stirred on the hot plate for 30 min until

the evaporation ultimately occurred, and the solution became viscous. Then, the mixture

will be poured and spread into a glass petri dish to produce a fibre-based plastic.

The bioplastic film will be dried for an hour at 50 °C the oven and then dried at

room temperature for two days approximately.

16
 9. Characterization of Film

9.1. Film Thickness

The thickness of film samples will be measured with a digital micrometer with an

accuracy of 1 µm. An average of 10 values will be used for further calculations.

9.2. Water Vapor Permeability (WVP) Measurements

The WVP of films will be measured gravimetrically adapted to edible materials.

Differential relative humidity (RH) (∆RH70) will be used by adding the distilled water

(100% RH) to the permeation cell (cup) and exposing it to 30% RH in a ventilated

climatic chamber, at 25 ± 1 ◦C. The water vapor permeability, WVP (g m −1s−1 Pa−1)

will be calculated at the steady state of the permeation process, i.e., at a constant

weight change of the cell, according to Equation:

where ∆m/∆t is the weight of moisture loss per unit of time (g s−1), A is the film area

exposed to the moisture transfer (9.08 × 10−4 m2 ), x is the film thickness (m), and ∆p

is the water vapor pressure difference between the two sides of the film.

9.3. Mechanical Properties

A tensile testing machine will be used to determine the tensile strength, the

Young’s modulus and the percentage of elongation at a breakpoint (E, %). The film

will be cut into a rectangular shape (1.5 × 5 cm). All samples will be equilibrated for 7

days at 50% of RH and at 25 ◦C. The equilibrated film will be clamped in the

extension grips of the testing machine and stretched at a rate of 50 mm min−1

until

17
rupture. The initial distance between film holders is 4 cm. TS, YM, and E will be plotted

by computer from the stress–strain curves.

9.4. Biodegradability of bioplastic

This test will be performed in Petri dishes containing a 3.9 g of potato dextrose

agar (PDA) dissolved in 100 mL distilled water. The samples (3×3 cm) will be placed

on the PDA medium and ±5 μL of Aspergillus niger will be dropped and spread on

each sample surface using a disposable sterile spreader. The Petri dishes will be

incubated at 37°C and 90% RH for 7 days. Microbial biodegradability will be

determined by observing the growth of microbes on the samples at an interval time

of 1 day.

9.5. Film Color

Film color will be measured with a colorimeter using the CIE-Lab color scale. The

following color parameters will be measured: L* (lightness), a* (redness) and b*

(yellowness). From the recorded parameters L*, a* and b*, the total color difference

∆E will be determined. It is defined that when ∆E is more than 3, the color change

can be perceived by human eye.

with L1, a1 and b1—for active film, L0, a0 and b0—control film. To evaluate the color

changes of the pH sensing films, the above procedure will be also used. Before

measurements, the film samples will be immersed in different pH (pH 2, 4, 5, 6, 7, 8,

10, and 12) and will be left for 10 min. After that, all samples will be collected and will

be evaluated. All color measurements will be taken at 5 different locations on the

surface of each film sample.

18
 9.6. FTIR Analysis

Fourier transform infrared (FTIR) spectroscopy will be carried out using an FTIR

spectrometer. FTIR spectra will be recorded in the frequency range from 4000

to 400 cm−1 using ATR (attenuated total reflectance) with a ZnSe crystal. For each

measurement, 64 scans will be taken with a resolution of 4 cm −1. The spectra will be

recorded in duplicate. The aim of this analysis is to determine the molecular level

modifications induced by the incorporation of BHA into the polymer chains.

10. Antioxidant Properties of Bioplastic

10.1. Total Phenolic Content

Total phenolic content (TPC) will be determined using the modified Folin-

Ciocalteu method. First, 100 µL of the appropriately diluted content will be mixed

with 200 µL of Folin–Ciocalteu reagent and 2 mL of distilled water. Then, 1 mL of

20% Na2CO3 (w/v) will be added and kept at 50 ◦C for 25 min. The absorbance

will be measured at 765 nm using a spectrophotometer. The solvent for extraction

will be used instead of fruit extract for blanks, and gallic acid (5 g L −1) will be used for

the analytical curve. TPC will be expressed as mg of gallic acid equivalent (GAE) g−1

of prepared film or mg of GAE g−1 of powdered extract.

10.2. Antioxidant Activity of the Films

The reducing capacity (antioxidant activity, AOA) will be determined using a

modified ferric reducing antioxidant power (FRAP) method. The procedure is as

follows: freshly prepared FRAP reagent will be mixed with 0.3 M acetate buffer (pH =

3.6), 2, 4, 6-tripyridyl-s-triazine (TPTZ) solution and 20 mM FeCl3·6H2O solution,

respectively, in a ratio of 10:1:1 (v/v/v) and incubated at 37 ◦C. The sample (film

or

19
 extract) and FRAP reagent will be mixed (300 µL and 2.25 mL, respectively) and will

be incubated for 10 min at 37 ◦C. The absorbance will be measured at 593 nm. The

blank sample will be prepared using either distilled water for extracts and aqueous

acetic acid for PHA-based samples. The obtained results will be expressed as the

mg of ascorbic acid equivalents (AAE) g−1 of the prepared films, or mg AAE g−1

of the extract.

11. Statistical Analysis

Statistical analysis will be performed using Xlstat-Pro (win) 7.5.3. All data will be

ranked and the statistical differences will be evaluated on the ranks using one-way

analysis of variance (ANOVA). In all cases, a value of p < 0.05 confidence level is

considered significant.

20
 BIBLIOGRAPHY

1. David Bodamer. “EPA’s Latest MSW Estimates.” Waste460, November 16, 2016.
https://www.waste360.com
2. Ramos, Marjaleen. “17.5 billion of plastics every year.” Manila Bulletin. January
2020. https://www.mb.com
3. Amar Mohanty, et al. “Review of recent advances in the biodegradability of
polyhydroxyalkanoate (PHA) bioplastics and their composites.” Green Chemistry.
August 17, 2020. https://pubs.rsc.org/en/content/articlelanding/2020/gc/d0gc01647k
4. Azmin Siti, et al. ‘Development and characterization of food packaging bioplastic film
from cocoa pod husk cellulose incorporated with sugarcane bagasse fiber.” Journal
of Bioresources and Bioproducts. November 2020.
https://www.sciencedirect.com/science/article/pii/S2369969820301146?via%3Dihub
5. Sundarraj Chockalingam, et al. “Bagasse Fiber – The Future Biocomposite Material:
A Review,” International Journal of ChemTech Research. January 20, 2014.
https://www.researchgate.net/publication/270453697_Bagasse_Fiber_-
_The_Future_Biocomposite_Material_A_Review
6. Fujita Shuzo, et al. “Function of Some Antioxidants as a Plasticizer for the
Biodegradable Plastic, Poly- (3-hydroxybutyrate-co-3-hydroxyvalerate) [P-(HB-HV)].”
Journal of Home Economics of Japan. January 21, 2021.
https://www.jstage.jst.go.jp/article/jhej1987/54/3/54_3_193/_pdf/-char/ja
7. F. Masood. “Polyhydroxyalkanoates in the Food Packaging Industry.”
Nanotechnology Applications in Food. 2017.
https://www.sciencedirect.com/science/article/pii/B978012811942600008X?via%3Di
hub
8. Amar Mohanty, et al. “Review of recent advances in the biodegradability of
polyhydroxyalkanoate (PHA) bioplastics and their composites.” Green Chemistry.
August 17, 2020. https://pubs.rsc.org/en/content/articlelanding/2020/gc/d0gc01647k
9. Ipsita Roy, et al. “Polyhydroxyalkanoates: bioplastics with a green agenda.” Current
Opinion in Microbiology. June 2010. https://doi.org/10.1016/j.mib.2010.02.006
10. Azmin Siti, et al. ‘Development and characterization of food packaging bioplastic film
from cocoa pod husk cellulose incorporated with sugarcane bagasse fiber.” Journal
of Bioresources and Bioproducts. November 2020.
https://www.sciencedirect.com/science/article/pii/S2369969820301146?via%3Dihub
11. Sundarraj Chockalingam, et al. “Bagasse Fiber – The Future Biocomposite Material:
A Review,” International Journal of ChemTech Research. January 20, 2014.
https://www.researchgate.net/publication/270453697_
12. Anne Helmenstine. “Chemistry of BHA and BHT Food Preservatives.” ThoughtCo.
July 30, 2019. https://www.thoughtco.com/bha-and-bht-food-preservatives-607393
13. Adanze Nwakaudu, et al. “The Use of Natural Antioxidant Active Polymer Packaging
Films for Food Preservation.” AppliedSignals Research & Publications. November 2,
2015. https://www.researchgate.net/publication/290821241/
14. Azrina Azlan, et al. “Anthocyanidins and anthocyanins: colored pigments as food,
pharmaceutical ingredients, and the potential health benefits.” Food and Nutrition
Research. August 13, 2017.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613902/

21
15. Muhammad Oka Ramadhan, et al. “Anthocyanins from Agro-waste as Time-
Temperature Indicator to Monitor Freshness of Fish Products.” Indonesian Journal of
Educational Research and Technology. May 17, 2021.
https://ejournal.upi.edu/index.php/AJSE/article/view/34228/14758
16. N H C Hamzah,et al. “Smart food packaging from sago starch incorporated with
anthocyanin from Brassica oleracea.” IOP Conf. Ser.: Earth Environ. 2021.
https://iopscience.iop.org/article/10.1088/1755- 1315/733/1/012030/
17. Ramon Catala, et al. “Advances in antioxidant active food packaging.” Trends in
Food Science & Technology. January 2014.
https://www.sciencedirect.com/science/article/abs/pii/S0924224413002288
18. Jonas Contiero, et al. “Polyhydroxyalkanoate Synthesis by Burkholderia glumae into
a Sustainable Sugarcane Biorefinery Concept.” Bioprocess Engineering. January 13,
2021. https://www.frontiersin.org/articles/10.3389/fbioe.2020.631284/ful

22
 RESEARCH PLAN

A. Rationale

Food packaging is a critical step in the food manufacturing process because it

protects the food from infection, maintains its quality, and extends its shelf life.

However, it is responsible for polluting our climate, blocking our drains and potentially

harming wildlife when it ends up in our oceans and rivers. Poor food packaging may

result in reducing its freshness and spoilage that leads to food waste. When compared

to petroleum-based alternatives, PHAs are more costly. Thus, previous studies

suggested that PHAs can be employed in biocomposite materials including bio-based

agro-residues to reduce costs while preserving performance in some applications. In

this study, the incorporation of sugarcane bagasse and butylated hydroxyanisole will be

analyze how these variables affects the physical and chemical, mechanical, thermal,

biodegradability, and surface of producing the Polyhydroxyalkanoates bioplastic. This

study aims to benefit the society, and it will reduce the food spoilages by giving a

warning to consumers about the conditions inside the food package through a color

changing food packaging.

B. 1. Research Questions

 Does the cellulose in Sugarcane Bagasse affect the color-changing

activity of Polyhydroxyalkanoates bioplastic?

 Does increasing the amount of butylated hydroxyanisole affect the

intensityof color changing of the food packaging?

23
2. Engineering Goals

 To produce a Polyhydroxyalkanoates bioplastic from sugarcane

bagasse and butylated hydroxyanisole.

 To determine the physico-chemical properties of sugarcane bagasse

and butylated hydroxyanisole for fabricating the

Polyhydroxyalkanoates bioplastic.

 To evaluate the color response of the Polyhydroxyalkanoates

bioplastic that is exposed to different pH.

3. Hypothesis

 If the cellulose of Sugarcane Bagasse has high antioxidant, then that

affects the color-changing activity of Polyhydroxyalkanoates

bioplastic.

 If the butylated hydroxyanisole is related to anthocyanin, then the

intensity of color-changing will increase and ensure the shelf-life of

foods inside the packaging.

4. Expected Outcome

The study's objectives will be attained to a quality that will allow for

future inquiry. Significant color changes in dry films as a function of pH will

be detected, indicating a promising approach for their usage as intelligent

sensors in food packaging. The researcher will produce an efficient

Polyhydroxyalkanoates bioplastic from sugarcane bagasse and butylated

hydroxyanisole for color-changing food packaging.

24
C. Procedures

1. Collection of Materials

1 kilogram of Sugarcane bagasse will be collected from a

sugarcane juice that was readily available from the Suki Market in Quezon City

for consumption while 100 ml of butylated hydroxyanisole will be purchased

from UNAHCO, Inc. in Mandaluyong. The other materials or chemicals will be

obtained from different laboratories in Metro Manila.

2. Isolation, selection and Optimization of bacterial growth of PHA-

producing bacteria from Sugarcane Bagasse

Bacterial will be isolated, will be collected, and will be purified from

sugarcane trash (e.g. Ralstoniaeutropha, Azospirillumbrasilense, Rhizobiumsp,

Bacillus sp) will be evaluated for their ability to produce bioplastics.

3. Production of PHA by selected isolates

Glucose and trace element solution will be autoclaved separately, and

reconstituted prior to inoculation. The culture will be prepared by sub

culturingthe isolates twice in nutrient broth. Then one ml of a 24 h old culture will

be inoculated into 100 mL production medium and will be incubated at 37 °C

and150 rpm for 48 h.

4. Measurement of Dry Biomass

For dry biomass measurement the culture will be centrifuged at

10,000 rpm for 15 min, and the pellet will be dried in an oven at 55 °C to

constant weight.

25
5. Extraction Process of PHA

The PHA will be extracted from the biomass via reflux by adding 25 mL of

dimethyl carbonate (DMC) as a solvent to different amounts of biomass in a

flask connected to a cooling column. After the extraction, vacuum filtration with

filter paper will be used to separate the biomass from the solution. A rotary

evaporator will be used to recover the solvent and to separate the PHA in the

form of a film attached to the wall of the flask. The obtained PHA will be left to

dry overnight.

6. Purification Process of PHA

The extracted PHA will be purified with 1-butanol via reflux. 20 mL of the

solvent will be added to different amounts of the polymer in a flask which will be

connected to a cooling column. The purification times tested were 1 h and 2 h.

The solvent will be separated from the jelly-like polymer by physically pressing it

out with a cloth. The 1-butanol will be separated from the solubilized

contaminants and recycled with a rotary evaporator. The experimental data will

be obtained under the different purification conditions were compared using first

an F-test for variances and then the adequate t-test for the p values.

7. Fibre extraction from sugarcane bagasse

100 g of fine bagasse powder will be treated with 1000 mL of NaOH

solution for an hour with constant stirring at room temperature. After that, the

treated bagasse solution will be washed and filtered using distilled water until a

neutral pH will be obtained. Then, 25 g of the dried fibre will be bleached by

stirring it with a 200 mL solution containing 1 mL of vinegar and 3 g of NaCl

26
 on the hot plate for two hours at 70 °C. This bleaching process will allow the

fibres to be less susceptible to UV and moisture, which will be considered as a

factor for the degradation process.

8. Production of Bioplastic Film

All of the ingredients consisting of 1.5 g of bagasse fibres, butylated

hydroxyanisole at concentrations of 3 wt. %, dissolved 50 mg PHA extract in

10 ml chloroform 1 mL of glycerin, 40 mL of distilled water, and 0.5 g sorbitol will

be prepared and will be blended in a 250 mL beaker. Then, the mixture will be

poured and spread into a glass petri dish to produce a fibre-based plastic. The

bioplastic film will be dried for an hour at 50 °C in the oven and then dried at

room temperature for two days approximately.

D. Risk and Safety

The researcher will undertake extraction and isolation of bacteria (e.g.

Aspergillus Niger, Ralstoniaeutropha, Azospirillumbrasilense, Rhizobiumsp, Bacillus

sp.) Wear gloves, lab clothing, face mask, and safety goggles when working in a

laboratory and the laboratory technician/assistant will help and supervise the

appropriate usage of the testing machines. The qualified scientist will advise the

researchers on the necessary techniques of Folin–Ciocalteu reagent and ferric

reducing antioxidant power method. Proper handling of 1-butanol, may cause skin

irritation, serious eye damage, and may cause respiratory irritation. Proper handling

of Dimethyl carbonate, is flammable. Digital micrometer, tensile testing machine,

and FTIR spectroscopy should be handled with care. 1-butanol, BHA, Glucose,

trace element solution, and solvent should not be tasted and smelled.

27
E. Data Analysis

1. Film Thickness

The thickness of film samples will be measured with a digital micrometer with

an accuracy of 1 µm. An average of 10 values will be used for further

calculations.

2. Water Vapor Permeability (WVP) Measurements

The water vapor permeability, WVP (g m −1s−1 Pa−1) will be calculated at the

steady state of the permeation process, i.e., at a constant weight change of the

cell, according to Equation:

where ∆m/∆t is the weight of moisture loss per unit of time (g s−1), A is the film

area exposed to the moisture transfer (9.08 × 10−4 m2 ), x is the film

thickness (m), and ∆p is the water vapor pressure difference between the two

sides of the film.

3. Mechanical Properties

A tensile testing machine will be used to determine the tensile strength, the

Young’s modulus and the percentage of elongation at a breakpoint (E, %). The

film will be cut into a rectangular shape (1.5 × 5 cm). The initial distance

between film holders is 4 cm. TS, YM, and E will be plotted by computer from

the stress–strain curves.

4. Biodegradability of bioplastic

This test will be performed in Petri dishes containing a 3.9 g of potato

dextrose agar (PDA) dissolved in 100 mL distilled water. The samples (3×3 cm)

will be placed on the PDA medium and ±5 μL of Aspergillus niger will be

28
 dropped and spread on each sample surface using a disposable sterile

spreader.

5. Film Color

Film color will be measured with a colorimeter using the CIE-Lab color scale.

The following color parameters will be measured: L* (lightness), a* (redness)

and b* (yellowness). From the recorded parameters L*, a* and b*, the total color

difference ∆E will be determined. It is defined that when ∆E is more than 3,

the color change can be perceived by human eye.

with L1, a1 and b1—for active film, L0, a0 and b0—control film. To evaluate the

color changes of the pH sensing films, the above procedure will be also used.

Before measurements, the film samples will be immersed in different pH (pH 2,

4, 5, 6, 7, 8, 10, and 12) and will be left for 10 min. All color measurements will

be taken at 5 different locations on the surface of each film sample.

6. FTIR Analysis

Fourier transform infrared (FTIR) spectroscopy will be carried out using an

FTIR spectrometer. FTIR spectra will be recorded in the frequency range from

4000 to 400 cm−1 using ATR (attenuated total reflectance) with a ZnSe

crystal. For each measurement, 64 scans will be taken with a resolution of 4

cm−1.

7. Total Phenolic Content

Total phenolic content (TPC) will be determined using the modified Folin- Ciocalteu

method. First, 100 µL of the appropriately diluted content will be mixed with 200

29
 µL of Folin–Ciocalteu reagent and 2 mL of distilled water. Then, 1 mL of 20%

Na2CO3 (w/v) will be added and kept at 50 ◦C for 25 min. The absorbance will be

measured at 765 nm using a spectrophotometer. TPC was expressed as mg of

gallic acid equivalent (GAE) g−1 of prepared film or mg of GAE g−1 of powdered

extract.

8. Antioxidant Activity of the Films

The reducing capacity (antioxidant activity, AOA) will be determined using a

modified ferric reducing antioxidant power (FRAP) method. Prepare FRAP reagent

with 0.3 M acetate buffer (pH = 3.6), 2, 4, 6-tripyridyl-s-triazine (TPTZ) solution and

20 mM FeCl3·6H2O solution. The absorbance will be measured at 593 nm. The

blank sample will be prepared using either distilled water for extracts and aqueous

acetic acid for PHA-based samples. The obtained results will be expressed as

the mg of ascorbic acid equivalents (AAE) g−1 of the prepared films, or mg AAE

g−1 of the extract.

9. Statistical Analysis

Statistical analysis was performed using Xlstat-Pro (win) 7.5.3. All data were ranked

and the statistical differences were evaluated on the ranks using one-way analysis of

variance (ANOVA).

F. Bibliography
1. Azmin Siti, et al. ‘Development and characterization of food packaging bioplastic

film from cocoa pod husk cellulose incorporated with sugarcane bagasse fiber.”

Journal of Bioresources and Bioproducts. November 2020.

https://www.sciencedirect.com/science/article/pii/S2369969820301146?via%3

2. Sundarraj Chockalingam, et al. “Bagasse Fiber – The Future Biocomposite

30
 Material: A Review,” International Journal of ChemTech Research. January 20,

2014. https://www.researchgate.net/publication/270453697_Bagasse_Fiber_-

3. Muhammad Oka Ramadhan, et al. “Anthocyanins from Agro-waste as Time-

Temperature Indicator to Monitor Freshness of Fish Products.” Indonesian

Journal of Educational Research and Technology. May 17, 2021.

https://ejournal.upi.edu/index.php/AJSE/article/view/34228/14758

4. Jonas Contiero, et al. “Polyhydroxyalkanoate Synthesis by Burkholderia glumae

into a Sustainable Sugarcane Biorefinery Concept.” Bioprocess Engineering.

January 13, 2021.

https://www.frontiersin.org/articles/10.3389/fbioe.2020.631284/full

5. Mia Kurek, et al. “Characterization of Food Packaging Films with Blackcurrant

Fruit Waste as a Source of Antioxidant and Color Sensing Intelligent Material.”

Molecules. April 28, 2021. https://www.mdpi.com/1420-3049/26/9/2569

31
 APPENDICES

CHAPTER 1
PLAGIARISM REPORT

CHAPTER 2
PLAGIARISM REPORT

CHAPTER 3
PLAGIARISM REPORT

32

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